Combination treatment with t-PA variant and low molecular weight heparin

ABSTRACT

The invention concerns an improved therapeutic regimen for the treatment of thrombolytic disorders, such as acute myocardial infarction (AMI). In particular, the present invention concerns the treatment of thrombolytic disorders, e.g., AMI, with a combination of a tissue plasminogen activator (t-PA) variant having improved fibrin specificity and extended plasma half-life when compared with wild-type human t-PA and a low molecular weight heparin.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/371,778, filed Feb. 21, 2003, which claims the benefit ofU.S. Provisional Patent Application Ser. No. 60/359,327, filed Feb. 22,2002, all of which applications are fully incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns an improved therapeutic regimen for thetreatment of thrombolytic disorders, such as acute myocardial infarction(AMI). In particular, the present invention concerns the treatment ofthrombolytic disorders, e.g., AMI with a combination of a tissueplasminogen activator (t-PA) variant having improved fibrin specificityand extended plasma half-life (when compared with wild-type human t-PA)and an anti-thrombin agent having anti-Xa and/or anti-IIa activity, suchas a low molecular weight heparin.

2. Description of the Related Art

Thrombolytic therapy has been a major advance in the treatment of acutemyocardial infarction (AMI). Thrombolytics can re-establish perfusion inoccluded arteries, resulting in smaller infarct size, improved leftventricular function, and improved short and long-term survival. See,e.g., Braunwald E., Circulation 79:441-1 (1989); Braunwald, N. Engl. J.Med. 329:1650-2 (1993); The GUSTO investigators. An internationalrandomized trial comparing four thrombolytic strategies for acutemyocardial infarction. N. Engl. J. Med. 329:673-82 (1993).

However, current regimens of thrombolytic-antithrombic therapy continueto be limited by failure of initial recanalization and reocclusion inabout 40-45% of patients (Cannon et al., J. Am. Col. Cardiol. 24:1602-10(1994); The GUSTO Angiographic Investigators, N. Engl. J. Med.329:1615-22 (1993); Cannon and Braunwald, Acta Cardiol. 49:1-8 (1994)).In addition, intracranial hemorrhage can occur in over 0.9% of thepatients treated with thrombolytics (ISIS-3 (Third International Studyof Infarct Survival) Collaborative Group, Lancet 339:753-70 (1992); TheGlobal Use of Strategies to Open Occluded Coronary Arteries (GUSTO) IIbInvestigators, N. Eng. J. Med. 335:775-82 (1996); ASSENT-IIInvestigators, Lancet 354:716-22 (1999)).

Attempts to increase the efficacy of thrombolytic therapy by increasingthe dose of thrombolytic have been unsuccessful because of unacceptablyhigh incidence of intracranial hemorrhage. Also, an important factor inreducing mortality associated with AMI is the reduction of time toreperfusion (Rawles, J., BMJ 312:212-15 (1996); Linderer et al., J. Am.Coll. Cardiol. 22:212-215 (1996); Weaver et al., JAMA 270:1211-16(1993)). Guidelines concerning the treatment of AMI have suggested atarget figure of 90 minutes for the maximum delay between the patientseeing help and receiving thrombolysis. Despite encouraging results,pre-hospital thrombolysis has not been widely implemented, and the delayfrom symptom onset to arrival to hospital, which constitutes about twothirds of the overall delay, remains a major impediment to optimalreperfusion. Accordingly, there is a great need for a thrombolyticregimen that allows early intervention, provides high efficacy, andcarries low risk of bleeding side-effects.

A particularly successful t-PA variant is tenecteplase (TNKase™), a t-PAvariant with extended half-life and improved fibrin specificity whencompared to native human t-PA. TNKase™ (TNK-t-PA; T103N, N117Q,KHRR(296-299)AAAA t-PA) is a 527 amino acids long glycoprotein developedby Genentech, Inc., which obtained FDA approval on Jun. 2, 2000 for usein the reduction of mortality associated with acute myocardialinfarction (AMI). TNKase™ (tenecteplase) is a derivative of wild-typehuman t-PA, which has a threonine (T) replaced by an asparagine at aminoacid position 103, adding a glycosylation site at that position, anasparagine (N) replaced by glutamine at position 117, removing aglycosylation site at that position, and four amino acids, lysine (K),histidine (H), arginine (R), and arginine (R) replaced by four alanines(A,A,A,A) at amino acid positions 296-299. A large scale clinical trial(ASSENT-II) has shown TNKase™ (tenecteplase) to be the optimalthrombolytic agent, due to its ease of single-bolus administration,equivalent mortality to t-PA, and lower bleeding risk, as a result ofits improved fibrin specificity (ASSENT-II Investigators, Lancet354:716-22 (1999)). While of all thrombolytic agents tested, TNKase™(tenecteplase) has been found to have the highest fibrin specificity,and its extended half-life allows single bolus dose administration,there is room for further improvement by way of improving efficacywithout increasing the risk of side-effects, such as intracranialbleeding and stroke. For safety of a single bolus administration oftenecteplase see also Van de Werf et al., Am. Heart J. 137:786-91(1999).

SUMMARY OF THE INVENTION

The present invention concerns an improved therapeutic regimen forfibrinolytic therapy. In particular, the invention concerns the combinedadministration of a t-PA variant suitable for single-dose administrationand an antithrombin agent having anti-Xa and/or anti-IIa activity, suchas a low molecular weight heparin.

Accordingly, in one aspect, the invention concerns a method comprisingadministering to a human patient in need of thrombolytic therapy aneffective amount of a combination of a human tissue plasminogenactivator (ht-PA) variant suitable for single-bolus administration, anda low molecular weight heparin.

In one embodiment, the ht-PA variant is glycosylated at any of positions103-105, and devoid of functional carbohydrate structure at position 117of wild-type ht-PA amino acid sequence, and exhibits a) extendedcirculatory half-life and substantially retained fibrin-binding, orimproved in vivo fibrinolytic potency, and b) improved fibrinspecificity, as compared to wild-type ht-PA. Preferably, the ht-PAvariant has extended circulatory half-life and substantially retainedfibrin binding as compared to wild-type ht-PA. In another preferredembodiment, the ht-PA variant has improved in vivo fibrinolytic potencyas compared to wild-type ht-PA.

One of the representative t-PA variants useful in the treatment methodof the present invention has an N-linked glycosylation at position 103of the wild-type ht-PA amino acid sequence. In a preferred t-PA variant,there is asparagine as part of an Asn-X-Ser or Asn-X-Thr tripeptidylsequence, wherein Asn is asparagine, Ser is serine, Thr is threonine,and X is any amino acid except proline, at position 103 of the wild-typeht-PA amino acid sequence. Another t-PA variant has asparagine atposition 103, tryptophane at position 104, and serine at position 105 ofthe wild-type ht-PA amino acid sequence.

ht-PA variants which additionally have an amino acid other thanasparagine at position 117 of the wild-type ht-PA amino acid sequencecan be advantageously used in the treatment methods herein. It isfurther of advantage to make further alterations within the wild-typeht-PA amino acid sequence in order to improve fibrin-specificity,relative to wild-type ht-PA. Such alterations preferably are in theserine protease domain of wild-type ht-PA, in particular within theamino acid region 296-302 or 274-277 of the wild-type ht-PA aminosequence. ht-PA variants having an alteration (preferably amino acidsubstitution) in the region 296-299 of the wild-type h-t-PA amino acidsequence are particularly preferred. In a specific variant, thealteration is the substitution of alanine for each of amino acidslysine, histidine, arginine, and arginine at respective positions 296,297, 298, and 299 of the wild-type ht-PA amino acid sequence.

The treatment method of the present invention can be carried out with avariety of low molecular weight heparin preparations, including, forexample, enoxaparin, dalteparin, tinzaparin, certoparin, pamaparin,nadroparin, ardeparin, and reviparin. A preferred low molecular weightheparin is enoxaparin.

Administration of the low molecular weight heparin and ht-PA variantshould take place as soon as possible following the onset of symptomsindicating that thrombolytic therapy is required. Thus, administrationshould preferably take place within about 8 hours, more preferablywithin about 6 hours, even more preferably within about 4 hoursfollowing the onset of symptoms.

The ht-PA variant and the low molecular weight heparin can beadministered simultaneously or in either order, following conventionalroutes of administration. In a specific embodiment, the low molecularweight heparin is administered first, followed by administration of theht-PA variant as a weight-adjusted single bolus.

The low molecular weight heparin, e.g., enoxaparin, is typicallyadministered as an intravenous bolus followed by subcutaneousadministration, where the subcutaneous administration may be repeated inperiodic intervals in order to reduce the likelihood of reocclusion.Thus, enoxaparin may be administered as an intravenous bolus of about 30mg immediately followed by a subcutaneous dose of about 1 mg/kg.

The condition to be treated can be any condition requiring thrombolytictherapy, such as, for example, myocardial infarction (MI), venousthrombosis, pulmonary embolism, cerebrovascular accident, and arterialembolism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the design of a randomized clinical trial (ASSENT-3)investigating the efficacy and safety of tenecteplase in combinationwith enoxaparin, abciximab, unfractionated heparin.

FIG. 2 shows the Kaplan-Meier curves for primary efficacy endpoint ofASSENT-3.

FIG. 3 shows the Kaplan-Meier curves for primary efficacy plus safetyendpoint of ASSENT-3.

FIG. 4 shows the relative risks and 95% CIs for primary efficacycomposite endpoint in the total ASSENT-3 study population and inprespecified subgroups.

FIG. 5 shows the relative risks and 95% CIs for primary efficacy plussafety composite endpoint in the total ASSENT-3 study population and inprespecified subgroups.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A. Definitions

The terms “wild-type human tissue plasminogen activator,” “wild-typehuman t-PA,” and “wild-type ht-PA” as used herein, refer to humanextrinsic (tissue-type) plasminogen activator having fibrinolyticactivity that typically has a structure with five domains (finger,growth factor, Kringle-1, Kringle-2, and protease domains). Thenucleotide and amino acid sequences of wild-type (native) human t-PAhave been reported by Pennica et al., Nature 301:214 (1983) and in U.S.Pat. No. 4,766,075, issued Aug. 23, 1988. The location of a particularamino acid in the polypeptide chain of t-PA is identified by a number.The number refers to the amino acid position in the amino acid sequenceof the mature, wild-type human t-PA polypeptide as disclosed in U.S.Pat. No. 4,766,075. In the present application, similarly positionedresidues in t-PA variants are designated by these numbers even thoughthe actual residue number is not so numbered due to deletions orinsertions in the molecule. This will occur, for example, withdeletional or insertional variants. The amino acids are identified usingthe one-letter or three-letter code. Substituted amino acids aresometimes designated herein by identifying the wild-type amino acid onthe left side of the number denoting the position in the polypeptidechain of that amino acid, and identifying the substituted amino acid onthe right side of the number. For example, replacement of the amino acidthreonine (T) by asparagine (N) at amino acid position 103 of thewild-type human t-PA molecule yields a t-PA variant designated T103Nt-PA. Similarly, the t-PA variant obtained by additional substitution ofglutamine (Q) for asparagine (N) at amino acid position 117 of thewild-type human t-PA molecule is designated T103N,N117Q t-PA. Deletionalvariants are identified by indicating the amino acid residue andposition at either end of the deletion, inclusive, and placing the Greekletter delta, “Δ”, to the left of the indicated amino acids. Insertionalt-PA variants are designated by the use of brackets “[ ]” around theinserted amino acids, and the location of the insertion is denoted byindicating the position of the amino acid on either side of theinsertion.

The various domains within the wild-type human t-PA (ht-PA) amino acidsequence have been designated, starting at the N-terminus of the aminoacid sequence of human tissue plasminogen activator, as: 1) the fingerregion (F) that has variously been defined as including amino acid 1upwards of about 44, 2) the growth factor region (G) that has beenvariously defined as stretching from about amino acid 45 upwards ofamino acid 91 (based upon its homology with EGF), 3) Kringle-1 (K1) thathas been defined as stretching from about amino acid 92 to about 173, 4)Kringle-2 (K2) that has been defined as stretching from about amino acid180 to about amino acid 261, and 5) the so-called (serine) proteasedomain (P) that generally has been defined as stretching from aboutamino acid 264 to the C-terminal end of the molecule. These domains aresituated contiguously generally of one another, or are separated byshort “linker” regions, and account for the entire amino acid sequencefrom about 1 to 527 amino acids in its putative mature form.

The term “human tissue plasminogen activator variant” or “ht-PA variant”is used to refer to a tissue plasminogen activator, which differs fromwild-type ht-PA at at least one amino acid position, and retains afunctional fibrin binding region and serine protease domain. The finger(F), growth factor (GF), and (to a lesser extent) Kringle-2 (K2) domainsof wild-type ht-PA are known to be involved in fibrin binding. An ht-PAvariant having a functional fibrin binding region will retain at leastthe minimal sequences of these domains that are required for fibrinbinding. The serum protease domain is responsible for the enzymaticactivity for wild-type ht-PA. An ht-PA variant having a functionalserine protease domain retains at least the minimal sequences from theserine protease domain of wild-type ht-PA required for convertingplasminogen to plasmin in the presence of a plasma clot or in thepresence of fibrin.

The terms “TNK t-PA,” “T103N, N117Q, KHRR(296-299)AAAA t-PA,”“tenecteplase,” and “TNKase™,” are used interchangeably and designate ahuman t-PA variant, which has a threonine (T) replaced by an asparagineat amino acid position 103, adding a glycosylation site at thatposition, an asparagine (N) replaced by glutamine at position 117,removing a glycosylation site at that position, and four amino acids,lysine (K), histidine (H), arginine (R), and arginine (R) replaced byfour alanines (A,A,A,A) at amino acid positions 296-299 of the wild-typehuman t-PA amino acid sequence. TNKase™ (Genentech, Inc., South SanFrancisco, Calif.) has been approved by the FDA for use in the reductionof mortality associated with AMI as a single intravenous bolus.

The terms “low molecular weight heparin” and “LMW heparin” are usedinterchangeably, and refer to heparin fractions typically prepared byfractionation and/or depolymerization of heparin so as to achievesignificant reduction in average molecular weight as compared with wholeheparin preparations. Compositions containing, procedures for making,and methods for using low molecular weight heparin are described invarious patent publications, including U.S. Pat. Nos. 4,281,108,4,687,765, 5,106,734, 4,977,250, 5,576,304, and EP 372 969, the contentsof which are hereby expressly incorporated by reference. LMW heparinsfor use in the present invention preferably have an average molecularweight of about 10 kD or less, more preferably of about 8 kD or less,most preferably less than about 5 kD. It is further preferred that LMWheparins should be of relatively uniform molecular weight e.g., with atleast about 60%, more preferably at least about 80% of polymer unitshaving a molecular weight within the above defined average molecularweight limits.

The terms “fibrin binding” and “fibrin binding affinity” refer to theability of the t-PA molecule to bind fibrin clots in standard fibrinbinding assays, such as the method described by Rijken et al., J. Biol.Chem. 257, 2920-2925 (1982) or its modified versions known in the art.

The terms “(t-PA) biological activity”, “biologically active”,“activity” and “active” refer to the ability of the t-PA molecule toconvert plasminogen to plasmin in the presence of a plasma clot or inthe presence of fibrin, as measured by the S-2288 assay, the plasma clotlysis assay, or other appropriate assays. The assay(s) may be conductedin the presence or absence of potential modulators of activity such asfibrin, fibrinogen, plasma and/or plasma clots.

The expressions “fibrinolytic activity,” “thrombolytic activity” and“clot lysis activity” are used interchangeably and refer to the abilityof a t-PA molecule to lyse a clot, whether derived from purified fibrinor from plasma, using any in vitro clot lysis assay known in the art,such as the purified clot lysis assay by Carlson, R. H. et al., Anal.Biochem. 168, 428-435 (1988) and its modified form described by Bennett,W. F. et al., J. Biol. Chem. 266 5191-5201 (1991).

The expressions “in vivo fibrinolytic potency”, in vivo thrombolyticpotency” and “in vivo clot lysis potency” are used interchangeably andrefer to clot lysis per unit dose of t-PA. “In vivo fibrinolyticpotency” is determined in any accepted animal model of clot lysis assay,including the hamster pulmonary embolism model (Collen, D. et al.,Thromb. Haemost. 65:174-180 (1991)), and the rabbit jugular veinthrombosis model (Collen, D. et al., J. Clin. Invest. 71:368-376(1983)).

The expression “substantially retain fibrin binding (affinity),”(compared to wild-type human t-PA) and grammatical variants thereof asused herein mean that the fibrin binding affinity (apparent K_(d) value)of the variant t-PA molecule is within about two fold of the fibrinbinding affinity (K_(d) value) for wild-type human t-PA as determined inthe same assay. The expression “substantially improved fibrin binding”refers to a greater than about four fold increase in the fibrin bindingaffinity (apparent K_(d) value) of a t-PA variant, as compared to thatof wild-type t-PA, caused by the inclusion of an additional mutation orset of mutations. The term “improved in vivo fibrinolytic potency”compared to wild-type t-PA refers to comparable in vivo clot lysisachieved by the administration of a variant t-PA at about one-third orless the dose of wild-type t-PA.

The terms “clearance rate” and “clearance” refer to the rate at whichthe t-PA molecule is removed from the bloodstream. Clearance (rate) ismeasured with respect to native t-PA, such that decreased clearance(rate) indicates that the t-PA variant is cleared more slowly thannative t-PA, and increased clearance (rate) indicates that the t-PAvariant is cleared more rapidly than native t-PA.

The term “circulatory half-life” means the half-life of a polypeptide ofinterest or polypeptide variant (e.g., an ht-PA variant) circulating inthe blood of a given mammal.

The expression “higher fibrin specificity” refers to the activity of at-PA variant that exhibits a higher ratio of fibrin-dependent specificactivity to fibrinogen-dependent specific activity in a S-2251 assay (ineither the one-chain or two-chain form) than wild-type rt-PA, andpreferably a ratio of at least about 1.5.

The expression “higher plasma clot specificity” refers to the activityof a t-PA variant that exhibits a higher ratio of plasma clot-dependentspecific activity to plasma-dependent specific activity in a S-2251assay (in either the one-chain or two-chain form) than wild-type rt-PA,and preferably a ratio of at least about 1.5.

The expression “devoid of functional carbohydrate structure at aminoacid position 117 of wild-type human t-PA” means complete removal of thecarbohydrate at amino acid residue 117, as where the glycosylationsignal is destroyed by site-directed mutagenesis, or substantialremoval, as by treatment with an endoglycosidase which may leave anintact N-acetylglucosamine residue linked to Asn 117, for example.

The term “thrombolytic disorder” is used in the broadest sense andrefers to any condition characterized by the formation of a thrombusthat obstructs vascular blood flow locally or detaches and embolizes toocclude blood flow downstream (thromboembolism). Thrombolytic disordersspecifically include, without limitation, myocardial infarction (MI),venous thrombosis, pulmonary embolism, cerebrovascular accident,arterial embolism, etc.

The term “myocardial infarction” or “MI” is used to refer to ischemicmyocardial necrosis usually resulting from abrupt reduction in coronaryblood flow to a segment of myocardium. MI is typically a disease of theleft ventricle (LV), but damage may extend to the right ventricle (RV)or atria.

The term “venous thrombosis” is used to include all forms of thrombosis,such as thrombosis affecting the superficial veins (superficialthrombophlebitis) and deep vein thrombosis (DVT). Since thrombosis isvirtually always accompanied by phlebitis, the terms “thrombosis” and“thrombophlebitis” are used interchangeably.

“Pulmonary embolism” is the sudden lodgment of a blood clot in apulmonary artery with subsequent obstructed blood supply to the lungparenchyma. The most common type of pulmonary embolus is a thrombus thatusually has migrated from a leg or pelvic vein. Most of those that causeserious hemodynamic disturbances form in an iliofemoral vein, either denovo or by propagation from calf vein thrombi. Thromboemboli originateinfrequently in the arm veins or in the right cardiac chambers.

The term “cerebrovascular accident” is used to refer to stroke and, ingeneral, infarction due to embolism or thrombosis of intracranial orextracarnial arteries, and associated hemorrhage.

“Coronary patency” is evaluated by angiography, using Thrombolysis InMyocardial Infarction (TIMI) criteria. The TIMI flow grades are definedas follows: TIMI flow 0=no perfusion; TIMI flow 1=penetration withoutperfusion; TIMI flow 2=partial perfusion with delayed run-off; TIMI flow3=complete perfusion with brisk run-off.

The term “antithrombotic therapy” refers to therapy aimed at preventingthe formation or growth of a blood clot, or partial or completedissolution of a blood clot already formed.

The terms “treat” or “treatment” refer to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to prevent,slow down (lessen), or reverse an undesired physiological change ordisorder, such as the formation of a blood clot and the development ofother physiological changes associated with the formation of bloodclots, e.g., restenosis; reocclusion; hemorrhage; hemodynamicdisturbances; pain, arrhytmias, sinus node disturbances,atrioventricular block, etc. associated with MI. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

As used herein, the phrase “effective amount” or “therapeuticallyeffective amount” is intended to include an amount of a compound orcombination of compounds, as applicable, to treat a thrombolyticdisorder in a mammal, including humans. The combination of compounds maybe, but does not have to be, a synergistic combination. “Synergy” asdescribed, for example, by Chou and Talalay, Adv. Enzyme Regul. 22:27-55(1984), occurs when the effect (in the present case the thrombolyticeffect) of the compounds when administered in combination is greaterthan the additive effect of the compounds when each is administeredalone, as a single agent.

The term “mammal” refers to any animal classified as a mammal, includinghumans, domestic and farm animals, and zoo, sports, or pet animals, suchas mouse, rat, rabbit, pig, sheep, goat, cattle and higher primates.

The terms “combination,” “combined” and similar expressions, when usedin reference to the administration of two or more compounds, mean thatthe compounds are administered to a subject concurrently. Concurrentadministration includes administration at the same time, in the sameformulation or separately, and sequential administration in any order orat different points in time so as to provide the desired therapeuticeffect.

B. Detailed Description

According to the World Health Organization, cardiovascular diseasesaccount for 12 million deaths in the world each year. Currently, heartattack is a leading killer of men and women in developed countries. Itis projected that by 2010, heart disease will be the number one cause ofdeath in developing countries.

In the last decade, thrombolytic therapy has emerged as the standard ofcare for the pharmacological management of thrombolytic diseases, suchas AMI. However, despite recent advances, most current thrombolyticregimens have significant shortcomings due to: (1) low optimalreperfusion rate (TIMI grade 3 flow in about 50% of patients treated),(2) time delay to reperfusion averaging 45-120 minutes, and (3)reocclusion of the infarct-related artery after initial successfulreperfusion in 5-10% of the patents treated and associated increasedmortality; and intracranial bleeding in up to 0.9% of patients treated.See, e.g., Jang et al., J. Am. Coll. Cardiol. 33:1879-85 (1999). Inorder to further improve treatment outcomes, cardiologists throughoutthe world are investigating various combinations of thrombolytic agentscombined with other pharmacological agents, such as anti-thrombotics andanti-platelet agents, in an attempt to maximize artery-opening patencyand reduce mortality.

The present invention concerns an improved therapy regimen for thetreatment of thrombolytic disorders, such as acute myocardial infarction(AMI). In particular, the invention is concerned with a new and improvedcombination therapy using a long half-life, fibrin specific t-PA variant(e.g., TNK t-PA) in combination with a low molecular weight heparin. Thenew treatment regimen combines improved efficacy and safety whencompared with other therapeutic approaches currently used in clinicalpractice.

B.1 Tissue Plasminogen Activator (t-PA) Variants

As discussed above, TNK t-PA (TNKase™, tenecteplase; T103N, N117Q,KHRR(296-299)AAAA t-PA) is a 527 amino acids long glycoprotein developedby Genentech, Inc., which obtained FDA approval on Jun. 2, 2000 for usein the reduction of mortality associated with acute myocardialinfarction (AMI). TNKase™ (tenecteplase) is a derivative of wild-typehuman t-PA, which has a threonine (T) replaced by an asparagine at aminoacid position 103, adding a glycosylation site at that position, anasparagine (N) replaced by glutamine at position 117, removing aglycosylation site at that position, and four amino acids, lysine (K),histidine (H), arginine (R), and arginine (R) replaced by four alanines(A,A,A,A) at amino acid positions 296-299. In the 113 patient TIMI 10Atrial, TNK t-PA was shown to have a prolonged half-life that allows itto be administered as a single intravenous (IV) bolus (Cannon et al.,Circulation 95:351-6 (1997)). Due to this property, TNK t-PA isparticularly suitable for pre-hospital administration.

In the TIMI 10B trial, an angiographic study with 886 patients, TNK t-PAwas demonstrated to have similar patency and TIMI flow as wild-typehuman t-PA. Wild-type t-PA was administered as a 15 mg bolus of t-PA, a0.75 mg/kg (up to 50 mg) infusion over 30 minutes, followed by 0.50mg/kg (up to 35 mg) infusion over 60 minutes. The 60-minute and90-minute TIMI flow data are shown in the following Tables 1 and 2.TABLE 1 60 min median 60 min TIMI flow 6- min TIMI corrected TIMIDrug/dose grade 2 and 3 flow grade 3 frame count (n) TNK t-PA 30 mg 78%48% 37 (N = 176) TNK t-PA 40 mg 79% 58% 37 (n = 84) TNK-t-PA 50 mg 79%45% 37 (n = 42) t-PA (n = 65) 78% 48% 40

TABLE 2 90 min 90 min TIMI flow 90 min TIMI corrected median Drug/dosegrade 2 and 3 flow grade 3 TIMI frame count TNK t-PA 30 mg 76% 55% 38 (n= 297) TNK t-PA 40 mg 79% 64% 30 (n = 131) TNK t-PA 50 mg 88% 66% 34 (n= 42) t-PA (n = 303) 82% 63% 33

In addition, of all thrombolytic agents tested in large clinical trials,TNK t-PA has the highest fibrin specificity. In the TIMI 10A trial,systemic fibrinogen and plasminogen levels in patients treated with TNKt-PA fell by only 5-10% at 1 and 3 hours following administration(Tanswell et al., J. Am. Coll. Cardiol. 19:1071-5 (1992)).

Based on its uniquely advantageous properties, such as easyadministration as a single rapid bolus, equivalence compared towild-type t-PA with respect to mortality and intracranial hemorrhage,and lower rates of major bleeding side-effects, TNK t-PA is a newstandard for thrombolytic therapy.

Although the invention will be illustrated by data obtained with TNKt-PA, it is contemplated that other ht-PA variants with similarproperties can be used in the treatment methods herein. In general, theuse of any ht-PA variant suitable for single bolus administration isspecifically within the scope of the present invention. The relativelyrapid clearance of wild-type ht-PA from the plasma, while it is anadvantage in patients needing emergency intervention after an eventinvolving thrombus formation, e.g., MI, requires continuous intravenousadministration to maintain therapeutic levels of t-PA in the bloodstream. Variants of wild-type ht-PA that are suitable for single bolusadministration are required to have extended circulatory half-life(decreased clearance) compared to wild-type ht-PA. In addition, it isdesirable that such variants have improved fibrin-specificity relativeto wild-type ht-PA in order to reduce the likelihood of bleedingincidence associated with bolus administration. ht-PA variants meetingthese requirements are disclosed, for example, in EP 643,772, the entiredisclosure of which is hereby expressly incorporated by reference.

An exemplary ht-PA variant developed for single-bolus administration islanoteplase (nPA), a deletion variant lacking the finger and epidermalgrowth factor domains of wild-type ht-PA (The TIME-II Investigators,Eur. Heart J. 21:2005-2013 (2000)). This ht-PA variant, developed byGenetics Institute and licensed to Bristol-Myers Squibb and Suntory, hasa longer circulatory half-life than wild-type ht-PA but is less fibrinspecific. See also Umemura et al., Eur. J. Pharmacol. 262:27-31 (1994).The use of lanoteplase is specifically within the scope herein. Othert-PA variants developed for bolus administration and having longercirculatory half-lives than wild-type ht-PA include monteplase (Kawai etal., J. Am. Col. Cardiol. 29:1447-1453 (1997)), in which Cys84 ofwild-type ht-PA is substituted by Ser, pamiteplase (Kawasaki et al.,Drug. Dev. Res. 33:33-38 (1994)), having amino acids 92 to 173 ofwild-type ht-PA deleted, an Arg275 substituted by Glu, and a truncatedt-PA variant D-K2P (Saito et al., Biotechnol. Prog. 10:472-479 (1994)),missing the finger and EGF domains.

Further exemplary ht-PA variants suitable for use in the combinationtherapy of the present invention are glycosylated at any of positions103-105 and lack functional carbohydrate structure at amino acidposition 117 of wild-type ht-PA. The removal of the functionalcarbohydrate structure can be accomplished by any method known in theart, but preferably is achieved by amino acid substitution for at leastone residue in the Asn-Ser-Ser glycosylation signal at positions 117-119of the wild-type ht-PA amino acid sequence. In a particularly relevantvariant, asparagine at amino acid position 117 is replaced by anotheramino acid, the preferred substituent being glutamine (see, also EP238,304; WO 89/04368; and U.S. Pat. No. 5,612,029). In addition, thecarbohydrate structure at amino acid position 117 of wild-type ht-PA canbe substantially removed by the use of an endoglycosidase, such asEndoglycolidase H (Endo-H). Endo-H is only capable of removal of highmannose and hybrid oligosaccharides. Accordingly, under appropriateconditions, Endo-H is capable of substantially removing the high mannosecarbohydrate structure at amino acid residue 117 of wild-type ht-PA,without functionally affecting the complex structures at amino acidresidues 184 and 448.

Glycosylation in the 103-105 amino acid region is preferablyaccomplished by creating an N-linked glycosylation signal at any ofthese positions. In particular, such variants will have asparagines aspart of an Asn-X-Ser, or Asn-X-Thr tripeptidyl sequence (X is any aminoacid other than proline) at position 103, 104, or 105 of the wild-typeht-PA amino acid sequence. The added N-linked glycosylation preferablyis at position 103. Such ht-PA variants will have an asparagine atposition 103, tryptophan at position 104, and serine at position 105 ofthe wild-type ht-PA amino acid sequence.

As discussed above, it is particularly desirable to use in thecombination therapy herein t-PA variants that are more fibrin-specificthan wild-type ht-PA. Such ht-PA variants will act more preferentiallyat the site of the fibrin clot than wild-type ht-PA and are, therefore,expected to cause diminished side-effects associated with the activationof circulating plasminogen, e.g., less severe and less frequent bleedingcomplications. Improved fibrin specificity can be achieved by analteration known in the art. Fibrin specificity can be improved, forexample, by amino acid alterations within the serine protease domain ofwild-type ht-PA. The amino acid alterations may be, for example,substitutions at one or more of amino acid positions 296-302, preferably296-299 of the serine protease domain. In a preferred variant, each ofthe amino acids lysine, histidine, arginine, arginine at positions296-299 of wild-type ht-PA is replaced by alanine. In a furtherpreferred variant, the arginines at positions 298 and 299 are bothreplaced by glutamic acid. In another preferred variant, the lysine,histidine, and proline at amino acid positions 296, 297 and 302 ofwild-type ht-PA are additionally replaced by glutamine, asparagine, andserine, respectively. In a further fibrin-specific variant,phenylalanine, arginine, isoleucine, and lysine at amino acid positions274, 275, 276 and 277 of the wild-type ht-PA amino acid sequence arereplaced by amino acids leucine, histidine, serine, and threonine,respectively. The latter alteration results in a loss of plasmincleavage site, therefore, the variants are substantially in a singlechain form.

In addition to the alterations resulting in enhanced circulatoryhalf-life and/or increased fibrin-specificity relative to wild-typeht-PA, the t-PA variants may contain additional amino acid alterations,e.g., substitutions, insertions and/or deletions to further improvetheir therapeutic properties.

Examples of suitable t-PA variants for use in accordance with thepresent invention include T103N, N117Y t-PA; S105N, A107S, N117Z t-PA;S105N, A107S, N117Z t-PA; T103N, N117Z, KHRR(296-299)AAAA t-PA; S105N,A107S, N117Z KHRR(296-299)AAAA t-PA; T103N, N117Z, R298E, R299E t-PA;S105N, A107S, N117Z, R298E, R299E t-PA; T103N, Ni 17Z, K296Q, H297N,P301S t-PA; S105N, N117Z, K296Q, H297N, P301S t-PA; T103N, N117Z,FRIK(274-277)LHST t-PA; S105N, A107S, N117Z, FRIK(274-277)LHST t-PA,wherein Z denotes any of the 20 naturally occurring amino acids, exceptasparagine (N). Particularly preferred are the ht-PA variants in whichat position 117 asparagine (N) is substituted by glutamine (Q).

Tenecteplase is commercially available from Genentech, Inc., South SanFrancisco, Calif. The other ht-PA variants can be readily prepared bywell known techniques of recombinant DNA technology and/or chemicalsynthesis.

The plasma clearance and fibrin specificity of the t-PA variants can betested, for example, as described by Paoni et al., Thrombosis andHaemostasis 70:307-312 (1993); Keyt et al., Proc. Natl. Acad. Sci. USA91:3670-3674 (1994); Refino et al., Thrombosis and Haemostasis70:313-319 (1993); or using a rabbit thrombosed carotid artery modeldescribed by Benedict et al., Circulation 92:3032-3040 (1995). Plasmaclearance, fibrin binding, and clot lysis activity can also be tested asdescribed in EP 643,772. The results obtained can then be confirmed inhuman clinical trials, similar to those conducted for tenecteplase, suchas, for example, the ASSENT-II study reported in, Lancet 354:716-22(1999).

B.2 Low Molecular Weight Heparin

Antithrombin therapy for patients receiving thrombolytics is believed tobe important to both inhibit thrombin that is already present prior tothrombolysis and that which is generated as a consequence ofadministration of a thrombolytic. Thrombin is one of the main stimuliresponsible for platelet activation in the setting of thrombolysis andplays a central role in the pathogenesis or coronary rethrombosis.Although standard antithrombin therapy with intravenous unfractionatedheparin is able to inhibit thrombin activity, it is less effective inthe inhibition of thrombin regeneration. Around 50 to 60% of patientshas either suboptimal or inadequate anticoagulation with unfractionatedheparin during thrombolysis, and when more intensive unfractionatedheparin regimens were tested, an increased rate of hemorrhages,including intracerebral hemorrhages, were observed (Antman, E. M.,Circulation 90:1624-30 (1994); The Global Use of Strategies to OpenOccluded Coronary Arteries (GUSTO) II a Investigators, Circulation90:1631-7 (1994)).

Low molecular weight heparins (LMWHs) are obtained from standardunfractionated heparin (UFH), and have been used for the prophylaxis andtreatment of venous thromboembolism (see, e.g., Schafer, A. I., HospitalPractice Jan. 15, 1997, pp. 99-106). LMWHs have also be used in thetreatment of unstable angina and non-Q wave myocardial infarction.Commercially available low molecular weight heparins include, forexample LOVENOX® (enoxaparin sodium injection, available from AventisPharma Inc. (Bridgewater, N.J.), described in U.S. Pat. No. 5,389,618),FRAGMIN™ (dalteparin sodium injection, available from Pharmacia, Inc.(Columbus, Ohio)), INNOHEP® (tinzaparin sodium, available from DuPontPharmaceuticals Company (Wilmington, Del.)), ALPHAPARIN™ (certoparin,available from Alpha, U.K.), FRAXIPAINE™(nadroparin calcium, availablefrom Sanofi-Synthelabo Canada, Inc.), NORMIFLO™ (ardeparin, availablefrom Wyeth Laboratories, U.S.), and CLIVARINE™ (reviparin sodium,available from ICN Pharmaceuticals).

A particularly advantageous low molecular weight heparin preparation isLOVENOX® (enoxaparin sodium injection), hereinafter referred to as“enoxaparin.” Enoxaparin is a low molecular weight heparin produced bydepolymerization of standard unfractionated heparin (UFH). Unlikeporcine UFH, which has a molecular weight of 12,000 to 15,000 Daltons,enoxaparin has an average molecular weight of 4,500 Daltons. Compared toUFH, it has more predictable pharmacokinetics, and a higher ratio ofanti-Factor Xa to anti-Factor IIa activity. Enoxaparin is also resistantto inactivation by platelet factor 4. In studies examining enoxaparin inacute coronary syndrome patients, enoxaparin has been shown to be safeand more effective than unfractionated heparin at reducing coronaryevents (Cohen et al., N. Engl. J. Med. 337:447-52 (1997); Antman. E. M.and Women's Hosp., Boston, Mass., Supplement to Circulation 17:504-2649(1998)).

B.3 Combination Therapy

The ht-PA variants and low molecular weight heparin used herein can beformulated according to known methods to prepare pharmaceuticalcompositions, whereby the active ingredient is combined with apharmaceutically acceptable carrier. The ht-PA variant formulationsinclude sterile aqueous solutions and sterile hydratable powders, suchas lyophilized formulations. Typically, an appropriate amount of apharmaceutically acceptable salt is also used in the formulation torender the formulation isotonic. A buffer, such as arginine base, incombination with phosphoric acid is also typically included at anappropriate concentration to maintain a suitable pH, generally fromabout 5.5 to about 7.5. In addition or alternatively, a compound such asglycerol may be included in the formulation to help maintain theshelf-life.

Tenecteplase is currently marketed as a sterile, white to off-white,lyophilized powder for single IV administration after reconstitutionwith Sterile Water for Injection (SWFI). Each vial of the commercialformulation of tenecteplase (TNKase™) nominally contains 52.5 mgtenecteplase, 0.55 g L-arginine, 0.17 g phosphoric acid, and 4.3 mgpolysorbate 20, which includes a 5% overfill, and each vial delivers 50mg of tenecteplase. The reconstituted solution contains 5 mg/mltenecteplase. However, other pharmaceutical formulations are alsospecifically within the scope of the present invention.

The t-PA variants of the present invention are typically administered asa single (occasionally repeated) intravenous (IV) bolus, in combinationwith a low molecular weight heparin, which is typically administeredintravenously and/or subcutaneously (SC). Bolus administration hasseveral advantages. The ease of administration facilitates earlyintervention and may make more feasible prehospital treatment withthrombolysis, and the simplicity of dosing may reduce medication errors,thereby reducing associated mortality.

In the treatment of thrombolytic disorders, such as MI, the t-PAvariant, such as TNK t-PA (TNKase™, tenecteplase), hereinafter referredto as “tenecteplase,” is preferably administered as a single intravenous(IV) bolus, in combination with a low molecule weight heparin, such asLOVENOX® (enoxaparin sodium injection), hereinafter referred to as“enoxaparin.” A typical dose for a t-PA variant such as tenecteplase isbetween about 15 mg and about 50 mg, depending on the body weight of thepatient, although lower and higher doses are also envisioned. Theoptimal dose depends on factors like the thrombolytic disorder targeted,the patient's sex, age, overall physical condition, the severity of thedisease, and the like. The determination of the optimal dose is wellwithin the skill of an ordinary physician. In a typical situation, to apatient weighing between about 70 and 80 kg, a t-PA variant such astenecteplase is typically administered in a dose of about 40 mg, as asingle IV bolus over 5-10 seconds, preferably over 5 seconds. For moreprecise, weight-adjusted dosing of the t-PA variant, see also Gibson etal., Am. J. Cardiol. 84:976-980 (1999). Weight adjusted dose typicallyvaries between about 0.2 mg/kg of body weight and about 1.25 mg/kg ofbody weight.

Low molecular weight heparin, such as enoxaparin, is typicallyadministered in conjunction with the t-PA variant, such as tenecteplase,starting with a fixed IV bolus followed by weight adjusted subcutaneous(SC) injections. For example, the low molecular weight heparin such asenoxaparin can be administered as a single IV bolus, followed by SCinjections. In a typical dosing regimen, a single IV bolus of about 30mg low molecular weight heparin (e.g., enoxaparin) is followed by about1 mg/kg SC injections about every 12 hours. In a typical situation, thefirst SC injection immediately follows the IV bolus, and treatmentfollows until the patient's discharge, or until revascularization, orfor 7 days, whichever comes first. Just as with the t-PA variant, e.g.,tenecteplase, the IV and SC doses of the low molecular weight heparin(e.g., enoxaparin) are determined to match the targeted thrombolyticdisorder, the patient's age, sex, overall physical condition, weight,and the like.

More specifically, patients are typically given enoxaparin at about 30mg if their body weight is less than about 60 kg, about 35 mg if it isabout 60-69 kg, about 40 mg if it is about 70-79 kg, about 45 mg if itis about 80-89 kg, and about 50 mg if it is about 90 kg or more.

In a large-scale clinical study (ASSENT-3), enrolling 6,095 heart attackpatients at more than 500 sites worldwide, details of which are providedin the Examples below, combination treatment with tenecteplase andenoxaparin resulted not only in improved clinical efficacy and safetybenefits, but also yielded an exceptionally low 30-day mortality rate(5.35%). This is the lowest reported mortality rate reported to date ina large-scale clinical trial of acute myocardial infarction.

Thrombolytic therapy with a combination of an ht-PA variant and a lowmolecular weight heparin in accordance with the present invention may becombined with the administration of aspirin as early as possiblefollowing the thrombotic event, and other therapeutic agents, such asβ-blockers, calcium channel blockers, angiotensin-converting enzyme(ACE) inhibitors, intravenous nitrates, β-blockers, angiotensin IIinhibitors, statins, ticlopidin/clopidogrel, oral anticoagulants,Abciximab, other gpIIb/IIIa inhibitors, angiotensin-receptor blockers,thienopyridines, and thrombolytics, all conventionally used in cardiactreatment.

Further details of the invention are provided in the followingnon-limiting example.

All literature and other citations throughout this application arehereby expressly incorporated by reference.

EXAMPLE

Efficacy and Safety of Tenecteplase in Combination with Enoxaparin inthe Treatment of Myocardial Infarction

Patients and Methods

Patients in 575 hospitals in 26 countries were recruited. Inclusioncriteria were identical to those of the Assessment of the Safety andEfficacy of a New Thrombolytic Regimen (ASSENT)-2 trial (Lancet354:716-22 (1999)), i.e., age 18 years or older, onset of symptoms lessthan 6 h before randomization, ST-segment elevation of at least 0.1 mVin two or more limb leads or at least 0.2 mV in two or more contiguouspericardial leads, or left bundle-branch block. Exclusion criteria onadmission were: systolic blood pressure of more than 180 mm Hg,diastolic blood pressure of more than 110 mm Hg, or both on repeatedmeasurements; use of abciximab or other glycoprotein IIb/IIIa inhibitorswithin the preceding 7 days; major surgery, biopsy of a parenchymalorgan or substantial trauma within 2 months; any head injury or othertrauma occurring after onset of current myocardial infarction; any knownhistory of stroke, transient ischemic attack, or dementia; any knownstructural damage to the central nervous system; current treatment withoral anticoagulants; treatment with unfractionated heparin of more than5000 U or a therapeutic subcutaneous dose of low-molecular-weightheparin within 6 h; known thrombocytopenia (<100 000 cells/μL); knownrenal insufficiency (serum creatinine concentration >221 μmol/L for menand >177 μmol/L for women); sustained cardiopulmonary resuscitation(more than 10 min) in previous 2 weeks; pregnancy, lactation, orparturition in the previous 30 days; active participation in anotherinvestigative drug or device study in the previous 30 days; previousenrollment in this study; any other disorder that would place thepatient at increased risk; and inability to follow the protocol and tocomply with the follow-up requirements.

The protocol was approved by each hospital's institutional review board,and patients gave informed consent.

Patients were randomly assigned, via a central computerized telephonesystem into one of the following three groups:

Group I: a bodyweight-adjusted single bolus of tenecteplase withenoxaparin (enoxaparin group).

Group II: low-dose tenecteplase, plus abciximab, plus low-doseunfractionated heparin (abciximab group).

Group III: full-dose tenecteplase with weight-adjusted unfractionatedheparin (heparin group).

Each patient was given a unique study number that corresponded with thenumber of a treatment kit. Study treatments were given on an open-labelbasis.

Tenecteplase was given over 5 seconds according to bodyweight: patientsassigned enoxaparin or unfractionated heparin were given 30 mg if theirbodyweight was less than 60 kg, 35 mg if it was 60-69 kg, 40 mg if itwas 70-79 kg, 45 mg if it was 80-89 kg, and 50 mg if it was 90 kg ormore. In patients assigned combination treatment with abciximab,half-dose tenecteplase was given with doses ranging from 15 mg to 25 mgaccording to the same weight categories as with the full dose.

Patients assigned weight-adjusted intravenous unfractionated heparinreceived a bolus of 60 U/kg (maximum of 4000 U) and initial infusion of12 U/kg per h (maximum 1000 U/h) adjusted to maintain an activatedpartial thromboplastin time of 50-70 seconds for 48 h with subsequentheparin administration left to the discretion of the treating physician.The first blood sample for activated partial thromboplastin timemeasurement was drawn after 3 h. Patients assigned enoxaparin co-therapyreceived an intravenous bolus of 30 mg immediately followed by the firstsubcutaneous dose of 1 mg/kg. To achieve sustained anticoagulation, thissubcutaneous dose was repeated every 12 h up to hospital discharge orrevascularization for a maximum of 7 days. The first two subcutaneousdoses could not exceed 100 mg. Patients assigned abciximab co-therapyreceived a 0·25 mg/kg bolus and 0·125 μg/kg per min (maximum of 10μg/min) for 12 h. Because abciximab has an anticoagulant effect, a lowerdose of unfractionated heparin was given: 40 U/kg bolus (maximum of 3000U) followed by 7 U/kg per h (maximum of 800 U/h) to achieve a partialthromboplastin time between 50 and 70 s. Also in this group, the firstactivated partial thromboplastin time was measured after 3 h. Aspirin(150-325 mg) was given to all patients. Intravenous boluses ofunfractionated heparin, enoxaparin, and abciximab were to be givenbefore bolus tenecteplase.

The primary endpoints were the composites of 30-day mortality,in-hospital reinfarction, or in-hospital refractory ischemia (primaryefficacy endpoint) to evaluate efficacy outcomes, and the above plusin-hospital intracranial hemorrhage or in-hospital major bleeding otherthan intracranial bleeding (primary efficacy plus safety endpoint) toevaluate efficacy improvements when safety adverse events were added tothe analysis.

Data were entered with the use of Oracle Clinical (version 3.1.1) andelectronically transferred to the central database in Leuven, Belgium.Safety data were reported monthly to the data and safety monitoringcommittee. All stroke cases were reviewed by the same stroke committeethat reviewed the stroke data in the ASSENT-2 trial. The members of thiscommittee were unaware of treatment assignment. There was no centraladjudication for the endpoints of reinfarction, refractory ischemia, andbleeding complications. However, definitions were provided to theinvestigators who, in addition, had to reconfirm the occurrence of theseendpoints on a special form.

Reinfarction in the first 18 h was defined as recurrent symptoms ofischemia at rest accompanied by new or recurrent ST-segment elevationsof 0.1 mV or more in at least two contiguous leads, lasting at least 30min. After 18 h, the definition was: new Q waves in two or more leads,or further increases in concentrations of creatine kinase MB, troponins,or total creatine kinase above the upper limit of normal and increasedover the previous value. Refractory ischemia was defined as symptoms ofischemia with ST-segment deviation or T-wave inversion persisting for atleast 10 min despite medical management and not fulfilling the diagnosisof reinfarction. Non-cerebral bleeding complications were defined asmajor (requiring transfusion, intervention because of hemodynamiccompromise, or both) or minor.

Statistical Analysis

Statistical analysis was by intention to treat. No confirmatorystatistical hypothesis was prespecified, but a detailed analysis planwas defined before the database was locked. This analysis plan was basedon generating risk ratios and CIs for the pairwise comparisons ofprimary interest. These comparisons were presented with the two-sided95% CI of the relative risk and with nominal p values. For the primaryendpoints, Kaplan-Meier curves were constructed and log-rank tests weredone. For each endpoint, a two-sided 95% CI was also calculated and anoverall χ test, comparing the three treatment groups, was done.Comparisons of interest were prespecified to first involve theunfractionated heparin and enoxaparin groups. If these were notdifferent, they were to be pooled and compared with the abciximab group.Otherwise, each experimental group was to be compared with theunfractionated heparin reference group.

On the basis of ASSENT-2, the estimated frequency of the primaryefficacy endpoint in the group allocated full-dose tenecteplase andunfractionated heparin was 13·8%. The frequency of the primary efficacyplus safety endpoint in this group was 17·7%. On the basis of phase-IIstudies, it was assumed that the two experimental groups would result ina better, or at least similar, outcome when compared with standardtreatment. The sample size and power calculations were therefore basedon non-inferiority of the two experimental groups versus the referencegroup. The study had 80% power to exclude, with 95% confidence(one-sided), a 1% higher rate of the primary endpoints compared with thereference group, provided the point estimate in the experimentaltreatment group was 1.7% lower for the efficacy endpoint and 2·0% lowerfor the efficacy plus safety endpoint.

Results

6095 patients were enrolled between May, 2000, and April, 2001, of whom5989 received study medication (FIG. 1). The baseline characteristicswere similar in the three groups. Overall, the study populations weresimilar to those of previous trials on thrombolytics. The time fromrandomization to bolus tenecteplase was significantly longer in theabciximab group because of the complexity of the treatments and the needto give the boluses of heparin and abciximab before the tenecteplasebolus. Concomitant medications given in hospital includedcalcium-channel blockers, intravenous nitrates, β-blockers, ACEinhibitors, angiotensin II inhibitors, statins, aspirin,ticlopidin/clopidogrel, oral anticoagulants, Abciximab, other gpIIb/IIIainhibitors, and thrombolytics, all conventionally used in cardiactreatment. High proportions of patients received β-blockers,angiotensin-converting-enzyme inhibitors, angiotensin-receptor blockers,statins, and thienopyridines. Abciximab and glycoprotein IIb/IIIainhibitors other than study medication were given most frequently in thegroups assigned full-dose tenecteplase and unfractionated heparin orenoxaparin. Likewise, low-molecular-weight heparins other than studymedication were most frequently given to patients assignedunfractionated heparin or abciximab.

The primary efficacy and efficacy plus safety endpoints and theirindividual components in the three treatment groups are shown in Table3. The combined efficacy and safety outcome in the full-dosetenecteplase plus unfractionated heparin group (Group III) of 17.0% wassimilar to that estimated (17.7%) before the trial commenced. TheKaplan-Meier curves for these primary endpoints are shown in FIGS. 2 and3. Log-rank tests were highly significant. Early after treatment, thecurves for the enoxaparin (Group III) and abciximab (Group II) groupsstarted to separate from that of unfractionated heparin (Group III). At48 h, the end of the unfractionated heparin infusion, differences in theprimary endpoints among the three groups were already present. For theprimary efficacy endpoint, event rates were 6.1% for full-dosetenecteplase plus enoxaparin, 5.2% for half-dose tenecteplase plusabciximab, and 8.8% for full-dose tenecteplase plus unfractionatedheparin (p<0.0001). For the primary efficacy plus safety endpoint, therates were 8.1, 8.2, and 10.3%, respectively (p=0.022). TABLE 3Frequency of composite and single endpoints at hospital discharge and at30 days Group I Group II Group III (n = 2040) (n = 2017) (n = 2038) p30-day mortality, 233/2037 223/2017 314/2038 0.0001 in-hospital rein-(11.4%) (11.1%) (15.4%) farction, or in- hospital refractory ischemia30-day mortality, 280/2037 287/2016 347/2036 0.0081 in-hospital rein-(13.8) (14.2%) (17.0%) farction, in- hospital refractory ischemia, in-hospital ICH, or in-hospital major bleeds (other than ICH) Death at 30days 109/2037 133/2017 122/2038 0.25  (5.4%)  (6.6%)  (6.0%) In-hospital54/2040  44/2017  86/2038 0.0009 reinfarction  (2.7%)  (2.2%)  (4.2%)In-hospital 93/2040  64/2017 132/2038 <0.0001 refractory ischemia (4.6%)  (3.2%)  (6.5%) In-hospital ICH 18/2040  19/2017  19/2038 0.98 (0.9%)  (0.9%)  (0.9%) Major bleeding 62/2040  87/2016  44/2035 0.0005(other than ICH)  (3.0%)  (4.3%)  (2/2%)ICH = intracranial hemorrhage

The relative risks in the total population and in the prespecifiedsubgroups are presented in FIGS. 4 and 5. The rates of the compositeendpoints were lower among patients treated with enoxaparin or abciximabthan among those treated with unfractionated heparin. Conventionalstatistical testing for full-dose tenecteplase plus enoxaparin versusfull-dose tenecteplase plus unfractionated heparin resulted in p valuesof 0·0002 and 0·0037, respectively, for the primary efficacy andefficacy plus safety composite endpoints. The half-dose tenecteplaseplus abciximab versus full-dose tenecteplase plus unfractionated heparincomparisons for the same primary endpoints yielded nominal p values of<0·0001 and 0·0142. After correcting for multiple testing (Bonferroni),conventional significance was reached for the primary efficacy endpointin the abciximab group (p=0.0002) but not for the efficacy plus safetyendpoint (p=0.057). For both the efficacy and efficacy plus safetyendpoints, statistical significance was reached in the enoxaparin group(p=0.0009 and p=0.0146, respectively).

The lower point estimate of the relative risk of the composite endpointswas consistent across subgroups for the combination of full-dosetenecteplase and enoxaparin. For the combination of half-dosetenecteplase and abciximab, lower point estimates were seen in mostsubgroups, except in patients older than 75 years and those withdiabetes (FIGS. 3 and 4). For the efficacy composite endpoint, the testfor an interaction between treatment and diabetes was significant(p=0.0004). For the efficacy plus safety composite endpoint, thetreatment interaction tests were significant for age (p=0.0010) anddiabetes (p=0.0007). In women, lower point estimates of the relativerisks of the efficacy composite endpoint were found in both experimentalgroups, whereas for the efficacy plus safety composite endpoint, thepoint estimates were on the line of unity.

No significant differences in 30-day mortality were present (Table 3).In-hospital reinfarction and refractory ischemia occurred lessfrequently in patients treated with enoxaparin or abciximab than inthose treated with unfractionated heparin. The rates of in-hospitaldeath or reinfarction were also lower in the enoxaparin and abciximabgroups than in the unfractionated heparin group: 138/2040 (6.8%) and148/2017 (7.3%) and 185/2038 (9.1%), respectively (p=0.0198). Nosignificant reductions in other major cardiac complications were seen,with the exception of a significantly lower need for urgent percutaneouscoronary interventions (ischemia-driven percutaneous coronaryintervention before hospital discharge) in patients on enoxaparin orabciximab than in patients on unfractionated heparin. Total in-hospitalstroke and intracranial hemorrhage rates were similar in the threegroups.

Non-cerebral bleeding complications, need for transfusion, and rates ofthrombocytopenia were also recorded. Significantly, more major bleedingcomplications (p=0.0002), more transfusions (p=0.001), and a higher rateof thrombocytopenia (p=0.0001) were seen in the abciximab group (GroupII) compared with the unfractionated heparin group (Group III). Inpatients older than 75 years and in diabetics, the rate of majorbleeding complications was three times higher with abciximab than withunfractionated heparin: 11/271 (4.1%) versus 31/233 (13.3%), and 8/363(2.2%) versus 25/355 (7.0%), respectively. More major bleedingcomplications and blood transfusions were also seen in the enoxaparingroup (Group I) compared with unfractionated heparin, although thesedifferences were not significant. There was no excess ofthrombocytopenia in this treatment group. The total number ofreadmissions to hospital was similar in the three treatment groups:254/1986 (12.8%) for enoxaparin, 221/1946 (11.4%) for abciximab, and239/1984 (12.1%) for unfractionated heparin (p==0.39). A few additionalstrokes occurred after hospital discharge in the three groups. Totalstroke rates and the rates of death or disabling stroke at 30 daysremained similar: 39/2040 (1.9%) and 122/2037 (6.0%) for full-dosetenecteplase and enoxaparin, 37/2017 (1.8%) and 141/2016 (7.0%) forhalf-dose tenecteplase and abciximab, and 34/2038 (1.7%) and 132/2038(6.5%) for full-dose tenecteplase and unfractionated heparin,respectively (p=0.83 for total stroke and p=0.43 for death or disablingstroke).

Discussion

The results of the group treated with full-dose tenecteplase andweight-adjusted unfractionated heparin (Group III) in this trial werevery similar to those of ASSENT-2. In ASSENT-2, a higher and not fullyweight-adjusted dose of unfractionated heparin was given and the firstpartial thromboplastin time was measured 6 h after start of treatment.Nonetheless, total mortality, reinfarction, total stroke, andintracranial hemorrhage rates were almost identical in both trials.However, there were fewer major bleeding complications (2.2% vs 4.7%)and less need for blood transfusion (2.3% vs 4.3%) in the present trialthan in ASSENT-2. These results indirectly support the use of a morefully weight-adjusted dose of unfractionated heparin, as recommended inthe ACC/AHA guidelines, together with earlier monitoring of the partialthromboplastin time. This unfractionated heparin dosing, however, wasnot associated with a reduction in the rate of intracranial hemorrhage,by contrast with the findings of a recent post-hoc analysis of theIntravenous tPA for the Treatment of Infarcting Myocardium Early(InTIME)-II data. Guigliano et al., Am. Heart J. 141:742-50 (2001).

Compared with unfractionated heparin, adjunctive therapy with abciximabor enoxaparin reduces ischemic complications of acute myocardialinfarction treated with tenecteplase. These reductions were found to bepresent early after the start of treatment. The results obtained withhalf-dose tenecteplase plus abciximab are very similar to those withhalf-dose reteplase and abciximab seen in GUSTO-V, and support thehypothesis that a more potent antiplatelet agent increases flow in theinfarct-related coronary artery. In both trials, these benefits areobtained at the cost of a higher rate of thrombocytopenia, majorbleeding complications, and blood transfusions. No benefit, and perhapseven harm, was seen in patients older than 75 years and in diabetics. Bycontrast with the present study, a 0.6% reduction in 30-day mortalitywas found in diabetic patients enrolled in GUSTO-V. Whether the findingsin diabetics from the smaller ASSENT-3 study described in this Exampleis due to chance or some other reason is unknown. Conversely, the datafrom both trials for this combination in elderly patients areconsistent. Taken together, they suggest that caution should beexercised regarding the use of conjunctive therapy with abciximab inelderly patients with an acute myocardial infarction treated with afibrinolytic agent. Further studies in the important and growingpopulation of elderly patients with an acute myocardial infarction arewarranted and might involve lower doses of these agents and mechanicalapproaches to reperfusion. The GUSTO-V and current results withhalf-dose fibrinolytic and abciximab suggest that there might be a rolefor this combination treatment in younger patients who are likely toundergo early coronary interventions. This speculation needs to beformally tested in future trials.

The reductions in ischemic complications in the full-dose tenecteplaseplus enoxaparin group were similar to those seen in the abciximab group,but were more consistent. Importantly, no increase in intracranialhemorrhage rate, no excess in thrombocytopenia, and only a modest andnon-significant increase in major bleeding complications was seendespite the length of treatment. In view of the present data and theease of administration, enoxaparin is regarded as an attractivealternative anticoagulant treatment when given in combination withtenecteplase.

The overall 30-day mortality rates in the present study were low andprobably result from selection of patients and an improvement inassociated medical treatment and intervention. However, time totreatment remained similar to that of other large trials of fibrinolytictherapy, emphasizing the opportunity provided by prehospital therapywith simple regimens. This opportunity is currently being explored inthe ASSENT-3 plus study which will compare the two full-dosetenecteplase cohorts administered out-of-hospital versus a matchedpopulation from the current study.

Like all clinical studies, the present study has some limitations.Ascertainment of selected components of the composite endpoints in thisopen trial was investigator-determined and subject to bias. The primarygoal was to examine whether addition of a low-molecular-weight heparinor a platelet glycoprotein IIb/IIIa inhibitor to a fibrinolytic agenthad promise as a therapeutic approach, and thus statistical hypotheseswere not defined a priori. Nonetheless, the strength and consistency ofthe results suggest that they are not due to bias or chance. Theobserved treatment effects with both experimental groups exceeded whatwas expected in this intermediate-sized trial and raises the question asto whether our exploratory experimental approach will be useful infuture assessments of promising combinations and various dosing regimensbefore large, definitive trials are done. The different duration ofheparin therapy in the enoxaparin versus the unfractionated heparingroup also deserves comment. We chose a 7-day course of enoxaparin toconform with previous studies in the hope of reducing recurrent ischemiccomplications and preventing reocclusion; the 48 h infusion ofunfractionated heparin is a standard antithrombotic strategy used inprevious trials such as ASSENT-2. The longer exposure to enoxaparinpossibly contributed to its increased efficacy and to the increasedtrend for bleeding.

Taking into account efficacy and safety, the combination of full-dosetenecteplase and long-term administration of enoxaparin emerged as thebest treatment, and the most promising reperfusion therapy regimen, inthis trial. This easy-to-administer therapy regimen lowered event rates,and exhibited an improved safety profile. Because of additionaladvantages such as the ease of administration and the lack of need formonitoring of anticoagulation, this combination should be regarded as anattractive alternative pharmacological reperfusion strategy.

Summary

In summary, while both Groups I and Group II improved the efficacy ofthe composite endpoint compared to Group III, only the tenecteplase plusenoxaparin arm (Group I) maintained this benefit when safety adverseevents were added to the analysis. Group I demonstrated the best resultsin reduction of events associated with the “primary efficacy plussafety” composite endpoint, including 30-day mortality, in-hospitalreinfarction or in-hospital ischemia, and reduction in in-hospital ICHor major bleeding complications.

While Group II (tenecteplase plus unfractionated heparin plus abciximab)did demonstrate an improvement in efficacy over Group III, Group II wasassociated with an increase in major bleeding complications includingnon-cerebral bleeding, need for transfusions and the occurrence ofthrombocytopenia, particularly in patients with diabetes or age 75 orolder, compared to Group I and Group III. Thus, Group I showed the bestresults in the “efficacy plus safety” analysis, with an event rate of13.75% compared with Group II (14.24%) and Group III (17.04%).

Generally similar across all three groups, the mortality rate, evaluatedas part of the combined efficacy plus safety endpoint, was lowest inGroup I, at 5.35%, compared to Group II at 6.59% and Group III at 5.99%.The differences were not statistically significant.

Although the present invention is illustrated with reference to certainembodiments, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and general knowledge in the art, andfall within the scope of the appended claims.

1. A method of selecting a patient for treatment with a combination of ahuman tissue plasminogen activator (ht-PA) variant suitable for singlebolus administration and low molecular weight heparin, comprisingselecting a patient for said treatment if (a) the patient has beendiagnosed with a thombolytic disorder; (b) the patient is 75 years ofage or younger; and (c) the patient is not diabetic.
 2. The method ofclaim 1 wherein said thrombolytic disorder is myocardial infarction(MI).
 3. The method of claim 2 further comprising the step of treatingsaid patient with said combination.
 4. The method of claim 3 whereinwherein said ht-PA variant is glycosylated at any of positions 103-105,and devoid of functional carbohydrate structure at position 117 ofwild-type ht-PA amino acid sequence, and exhibits a) extendedcirculatory half-life and fibrin-binding, affinity within about two-foldof the wild-type t-PA fibrin binding affinity or improved in vivofibrinolytic potency, and b) improved fibrin specificity, as compared towild-type ht-PA.
 5. The method of claim 4 wherein said ht-PA variant hasextended circulatory half-life and fibrin-binding affinity within abouttwo-fold of the wild-type t-PA fibrin binding affinity as compared towild-type ht-PA.
 6. The method of claim 4 wherein said ht-PA variant hasimproved in vivo fibrinolytic potency as compared to wild-type ht-PA. 7.The method of claim 4 wherein said ht-PA variant is glycosylated atposition 103 of the wild-type ht-PA amino acid sequence.
 8. The methodof claim 7 wherein said glycosylation is N-linked.
 9. The method ofclaim 8 wherein said ht-PA variant has asparagine as part of anAsn-X-Ser or Asn-X-Thr tripeptidyl sequence, wherein Asn is asparagine,Ser is serine, Thr is threonine, and X is any amino acid except proline,at position 103 of the wild-type ht-PA amino acid sequence.
 10. Themethod of claim 9 wherein said t-PA variant has asparagine at position103, tryptophan at position 104, and serine at position 105 of thewild-type ht-PA amino acid sequence.
 11. The method of claim 7 whereinsaid ht-PA variant has an amino acid other than asparagine at position117 of the wild-type ht-PA amino acid sequence.
 12. The method of claim7 wherein said ht-PA variant has an amino acid other than asparagine atposition 117 of the wild-type ht-PA amino acid sequence.
 13. The methodof claim 10 wherein said ht-PA variant has an amino acid other thanasparagine at position 117 of the wild-type ht-PA amino acid sequence.14. The method of claim 11 wherein said variant has improved fibrinspecificity as compared to wild-type h-tPA.
 15. The method of claim 14wherein said improved fibrin specificity is achieved by an alterationwithin the amino acid region 296-302 or 274-277 of the wild-type ht-PAamino sequence.
 16. The method of claim 15 wherein said alteration is inthe region 296-299 of the wild-type h-t-PA amino acid sequence.
 17. Themethod of claim 16 wherein said alteration is the substitution ofalanine for each of amino acids lysine, histidine, arginine, arginine atpositions 296, 297, 298, and 299 of the wild-type h-tPA amino acidsequence.
 18. The method of claim 3 wherein said low molecular weightheparin is selected from the group consisting of enoxaparin, dalteparin,tinzaparin, certoparin, pamaparin, nadroparin, ardeparin, and reviparin.19. The method of claim 18 wherein said low molecular weight heparin isenoxaparin.
 20. The method of claim 3 wherein said ht-PA variant isadministered intravenously as a single bolus dose.
 21. The method ofclaim 20 wherein said administration takes place within about 8 hoursfollowing the onset of symptoms requiring thrombolytic therapy.
 22. Themethod of claim 20 wherein the bolus dose is weight adjusted.
 23. Themethod of claim 22 wherein said bolus is administered within about 5seconds.
 24. The method of claim 23 wherein said t-PA variant istenecteplase.
 25. The method of claim 24 wherein said bolus dose isabout 30 mg for a patient having a bodyweight less than about 60 kg. 26.The method of claim 24 wherein said bolus dose is about 35 mg for apatient having a bodyweight of about 60 to 69 kg.
 27. The method ofclaim 24 wherein said bolus dose is about 40 mg for a patient having abodyweight of about 70 to 79 kg.
 28. The method of claim 24 wherein saidbolus dose is about 45 mg for a patient having a bodyweight of about 80to 89 kg.
 29. The method of claim 24 wherein said bolus dose is about 50mg for a patient having a bodyweight of about 90 kg or more.
 30. Themethod of claim 20 wherein administration of said low molecular weightheparin takes place before the single bolus administration of said ht-PAvariant.
 31. The method of claim 30 wherein said low molecular weightheparin is administered as an intravenous bolus followed by subcutaneousadministration.
 32. The method of claim 31 wherein said subcutaneousadministration is repeated.
 33. The method of claim 32 wherein saidsubcutaneous administration is repeated about every 12 hours for amaximum of about 7 days.
 34. The method of claim 30 wherein said lowmolecule weight heparin is enoxaparin.
 35. The method of claim 34wherein said enoxaparin is administered as an intravenous bolus of about30 mg immediately followed by a subcutaneous dose of about 1 mg/kg. 36.The method of claim 35 wherein said subcutaneous dose is repeated aboutevery 12 hours for a maximum of about 7 days.
 37. The method of claim 3wherein said patient is additionally administered aspirin.
 38. Themethod of claim 3 wherein said patient is monitored for at least 30 daysfollowing said administration.
 39. The method of claim 38 wherein saidpatient does not suffer reinfarction during the period of monitoring.40. The method of claim 38 wherein said patient is not diagnosed withrefractory ischemia during the period of monitoring.
 41. The method ofclaim 40 wherein said patient does not suffer intracranial hemorrhage orother major bleeding during the period of monitoring.